Midfielders Elano, of Brazil, and Deco, of Portugal, are likely to miss the clash between the two teams on Friday in Durban, DPA reported.
Elano's absence comes in addition to that of star playmaker Kaka, who is suspended after a red card.
The Galatasaray midfielder was kicked in the right ankle during the match against Ivory Coast on Sunday. Elano, who scored goals in Brazil's first two World Cup matches, returned to training on Wednesday but had to practice separately after feeling pain in his leg.
Deco suffered a hip injury in training prior to Portugal's second World Cup match, against North Korea on Monday, and has reportedly not fully recovered.
Deco is one of three Brazilian-born players in the Portugal squad, alongside defender Pepe and striker Liedson.
Brazil have already qualified for the Round of 16 in Group G. Portugal could still be eliminated in the first round, although their 7-0 win against North Korea gives them a comfortable goal difference and makes elimination unlikely.
Should Portugal beat Brazil, they could still end top of the group.